Illuminating the Dynamic Changes Associated with Heterotropic Allosteric Communication in Phosphofructokinase from Bacillus stearothermophilus

Abstract

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) undergoes dynamic changes in its otherwise rigid structure upon binding of ligands, fructose 6-phosphate (F6P) in the active sites, and allosteric inhibitor phosphoenolpyruvate (PEP) in the allosteric sites. PFK can exist in four distinct ligation states: Apo BsPFK, BsPFK-F6P, PEP-BsPFK, and PEP-BsPFK-F6P, however, the biophysical properties of each ligation state are poorly understood particularly with respect to their dynamic character. BsPFK is a homotetramer with four active sites and four allosteric sites, creating an intricate network of both homotropic and heterotropic interactions. The intrinsic tryptophan fluorescence emanating from hybrid enzymes in which a single tryptophan was used to report local side-chain dynamics upon ligand binding to sites involved in a single heterotropic interaction. Tryptophan was positioned by constructing conservative tryptophan-shift mutant enzymes in which one of the sixteen native phenylalanine and tyrosine residues in each subunit was substituted with tryptophan and the native tryptophan was changed to tyrosine. The first site of interest was a phenylalanine at position 230. Fluorescence intensity and steady-state anisotropy measurements reveal no change in response to F6P binding, but a 10% increase in intensity and a slight increase in steady-state anisotropy for PEP alone. Time-resolved experiments indicated that rotational correlation time significantly increased (by greater than 1 ns) for the PEP-BsPFK and PEP-BsPFK-F6P ligation states, but only slightly increased in the presence of F6P alone when compared to the unligated state of BsPFK. These trends are opposite comparable measurements made with PFK from E. coli (EcPFK). These results may suggest a structural basis for the observation that allosteric communication in BsPFK is entropically dominated whereas it is enthalpically dominated in EcPFK. Funding is provided by NIH grant GM33216 and Welch Foundation grant A1543.